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Covalently coupling the antibody on an amine-self-assembled gold surface to probe hyaluronan-binding protein with capacitance measurement.

Hyaluronan-binding proteins (HABPs), the important structural components of extracellular matrices, served important structural and regulatory functions during development and in maintaining adult tissue homestats. A sensitive, specific and rapid-responsing immunosensor to probe hyaluronan-binding cartilage protein was presented in this work. The novel immunosensor supplied a label-free detection method for HABP, which was based on measuring the capacitance change in-between the unlabeled HABP (antigen) and rabbit-anti-HABP (Ra-HABP, antibody). The HABP immunosensor was prepared by covalently coupling Ra-HABP on an amine-self-assembled gold surface with glutaraldehyde. The capacitance change corresponding to the concentration of HABP, the target antigen, was evaluated by an electrochemical approach called potentiostatic-step in microseconds. The immunosensor showed a specific response to HABP in the range 10-1000 ng/ml. The presented work supplied a promising clinical screening method.

Adsorption↗

Optimization of the gain-bandwidth product of capacitive micromachined ultrasonic transducers.

Capacitive micromachined ultrasonic transducers (cMUT) have large bandwidths, but they typically have low conversion efficiencies. This paper defines a performance measure in the form of a gain-bandwidth product and investigates the conditions in which this performance measure is maximized. A Mason model corrected with finite-element simulations is used for the purpose of optimizing parameters. There are different performance measures for transducers operating in transmit, receive, or pulse-echo modes. Basic parameters of the transducer are optimized for those operating modes. Optimized values for a cMUT with silicon nitride membrane and immersed in water are given. The effect of including an electrical matching network is considered. In particular, the effect of a shunt inductor in the gain-bandwidth product is investigated. Design tools are introduced, which are used to determine optimal dimensions of cMUTs with the specified frequency or gain response.

Computer Simulation↗

Differential effects of polyunsaturated fatty acids on membrane capacitance and exocytosis in rat pheochromocytoma-12 cells.

The fusion of synaptic vesicles with the plasma membrane during exocytosis can be recorded by membrane capacitance measurements under voltage-clamp conditions. These measurements enable high time-resolution quantitation of exocytosis. The present study was carried out using the above technique to elucidate the effects of various polyunsaturated fatty acids on exocytosis in a neuroendocrine cell, the rat pheochromocytoma-12 (PC12) cell. External application of eicosapentaenoic acid and arachidonic acid resulted in an increase in capacitance of PC12 cells, indicating fusion of secretory vesicles with cell membranes and exocytosis. In contrast, docosahexaenoic acid, linoleic acid, oleic acid, and vehicle control had no significant effect on capacitance. The above findings show differential effects of polyunsaturated fatty acids on exocytosis in PC12 cells. It is postulated that besides arachidonic acid, eicosapentaenoic acid could also play an important role in exocytosis and neurotransmitter release, in neurons and hormone-secreting cells.

Animals↗

Comparison of transepidermal water loss, capacitance and pH values in the skin between intrinsic and extrinsic atopic dermatitis patients.

Atopic dermatitis (AD), with the prevalence rate of around 10 to 15%, is characterized by an intensely pruritic skin lesions with typical distribution and morphology. Recently, AD is divided into extrinsic type (ADe) and intrinsic type (ADi) according to the laboratory findings and associated diseases. ADe is well-known for high IgE level, positive response to food- or aero-allergens, whereas ADi has clinically similar skin lesions and distribution patterns of AD with normal serum IgE levels, negative in vitro test for environmental or food allergens and without associated atopic diseases. To instrumentally evaluate the differences of skin involvement and functions between ADi and ADe, we checked the transepidermal water loss (TEWL), capacitance and pH in both types of childhood AD and age-matched control. The proportion of ADi was around 20% in all AD patients (10/51). Our experiment suggested possible differences between ADi and ADe. Antecubital fossa is a famous involvement site of childhood type of AD, where both types of AD patients showed higher TEWL and decreased capacitance. ADe patients showed increased TEWL in all sites and lower hydration in 4 sites, whereas ADi patients showed no significant differences of TEWL and hydration in forehead, cheek, and back of leg.

Adolescent↗

The effect of hydrostatic pressure on S-layer-supported lipid membranes.

We report on the behavior of unsupported and surface layer (S-layer)-supported lipid membranes at the application of a uniform hydrostatic pressure. At a hydrostatic pressure gradient higher than 6 N/m(2), unsupported lipid membranes, independent from which side pressurized and S-layer-supported lipid membranes pressurized from the lipid-faced side revealed a pronounced increase in capacitance. A maximal hydrostatic pressure gradient of 11.0 N/m(2) resulted in an almost doubling of the capacitance of the (composite) membranes. S-layer-supported lipid membranes showed a hysteresis in the capacitance versus pressure plot, indicating that this composite structure required a certain time to reorient when the pressure gradient acting from the lipid-faced side was balanced. By contrast, the S-layer-supported lipid membrane pressurized from the protein-faced side revealed only a minute increase in capacitance (C/C(0,max)=1.17+/-0.05), reflecting only minor pressure-induced area expansion. In addition, no hysteresis could be observed, indicating that no rearrangement of the composite membrane occurred. The maximal induced tension was with 4.3+/-0.2 mN/m, significantly higher than that of unsupported (2.5+/-0.3 mN/m) and S-layer-supported lipid membranes pressurized from the lipid-faced side (2.6+/-0.1 mN/m).

Bacterial Proteins↗

Capacitive sensing of droplets for microfluidic devices based on thermocapillary actuation.

The design and performance of a miniaturized coplanar capacitive sensor is presented whose electrode arrays can also function as resistive microheaters for thermocapillary actuation of liquid films and droplets. Optimal compromise between large capacitive signal and high spatial resolution is obtained for electrode widths comparable to the liquid film thickness measured, in agreement with supporting numerical simulations which include mutual capacitance effects. An interdigitated, variable width design, allowing for wider central electrodes, increases the capacitive signal for liquid structures with non-uniform height profiles. The capacitive resolution and time response of the current design is approximately 0.03 pF and 10 ms, respectively, which makes possible a number of sensing functions for nanoliter droplets. These include detection of droplet position, size, composition or percentage water uptake for hygroscopic liquids. Its rapid response time allows measurements of the rate of mass loss in evaporating droplets.

Capillary Action↗

Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells.

This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

Alternative Splicing↗

On-line biomass monitoring of CHO perfusion culture with scanning dielectric spectroscopy.

In this work, dielectric spectroscopy was used to monitor two CHO perfusion culture experiments (B14 and B16). The capacitance of the cell suspension was recorded every 20 minutes over an excitation frequency range of 0.2 MHz to 10.0 MHz. A phase plot of the capacitance at a low excitation frequency vs. the value at a higher frequency proved to be an accurate indicator of the major transition points of the culture, i.e., maximum cell viability, end of lactate consumption, point of zero viability. For both experiments, the capacitance signal correlated very well (R(2) >0.98) with viable cell number up to concentrations of 1 x 10(7) cells/mL. Visual observation of the capacitance spectra indicated that changes in the capacitance relative to frequency were related to the cellular morphology. A multivariate model was developed using off-line data that could predict the median cell diameter within a single experiment (B14) with an error of 0.34 microm (2%). Upon extension to a subsequent experiment (B16), the predicted error was 1.18 microm (9%).

Algorithms↗

Responsive corneosurfametry following in vivo skin preconditioning.

Skin is subjected to many environmental threats, some of which altering the structure and function of the stratum corneum. Among them, surfactants are recognized factors that may influence irritant contact dermatitis. The present study was conducted to compare the variations in skin capacitance and corneosurfametry (CSM) reactivity before and after skin exposure to repeated subclinical injuries by 2 hand dishwashing liquids. A forearm immersion test was performed on 30 healthy volunteers. 2 daily soak sessions were performed for 5 days. At inclusion and the day following the last soak session, skin capacitance was measured and cyanoacrylate skin-surface strippings were harvested. The latter specimens were used for the ex vivo microwave CSM. Both types of assessments clearly differentiated the 2 hand dishwashing liquids. The forearm immersion test allowed the discriminant sensitivity of CSM to increase. Intact skin capacitance did not predict CSM data. By contrast, a significant correlation was found between the post-test conductance and the corresponding CSM data. In conclusion, a forearm immersion test under realistic conditions can discriminate the irritation potential between surfactant-based products by measuring skin conductance and performing CSM. In vivo skin preconditioning by surfactants increases CSM sensitivity to the same surfactants.

Adult↗

Capacitance measurements of exocytosis in mouse pancreatic alpha-, beta- and delta-cells within intact islets of Langerhans.

Capacitance measurements of exocytosis were applied to functionally identified alpha-, beta- and delta-cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in beta-cells during a depolarization to 0 mV was equivalent to 14 granules s(-1), <5% of that observed in isolated beta-cells. Beta-cell secretion exhibited bell-shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to approximately -20 mV) the maximum rate of release was approximately 4 granules s(-1). Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L-type Ca(2+) channel blocker nifedipine (25 microm) but only marginally (<20%) affected by the R-type Ca(2+) channel blocker SNX482 (100 nm). Exocytosis in the glucagon-producing alpha-cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s(-1)) and a sustained late component (30 granules s(-1)). Whereas addition of the N-type Ca(2+) channel blocker omega-conotoxin GVIA (0.1 microm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L-type Ca(2+) channel blocker nifedipine (25 microm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca(2+)-influx through N-type rather than L-type Ca(2+) channels. Exocytosis in the somatostatin-secreting delta-cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s(-1)) component followed by a sustained slower (60 granules s(-1)) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in beta-cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

Action Potentials↗

Time response and stability of porous silicon capacitive immunosensors.

The time response of affinity sensors made with nanostructured materials is a topic of considerable interest, since affinity sensors made with nanostructured materials provide greater sensitivities than corresponding planar crystalline devices but at the cost of stability and drift. We present a study of the time response of capacitive immunosensors made using porous silicon and ultrathin room temperature anodic oxide. It was found that sensor drift can be substantial but can be reduced by subjecting the capacitive immunosensor in buffer to an anodic bias that is larger than the bias at which sensor capacitance is measured. By measuring sensor response before the addition of the analyte and using it for baseline correction after addition of the analyte, the effect of nonspecific sensor drift can be further reduced. We observed that after the addition of the analyte to the porous silicon immunocapacitor, there is a fast decrease in capacitance (order of tens of seconds) followed by a slow increase (order of tens of minutes), which models well as a sum of exponents with a fast exponential decay followed by a slow exponential rise. Possible processes that can give rise to such a response are perturbations of the double layer for the fast decay and column resistance switching for the slow rise.

Biosensing Techniques↗

Monitoring the interfacial capacitance at self-assembled phosphate monolayers on gold electrodes upon interaction with calcium and magnesium.

Electrochemical impedance spectroscopy has been used to evaluate the change in interfacial capacitance upon calcium and magnesium coordination to a phosphate-modified electrode. The phosphate electrode was prepared via immobilization of phosphorylated, thiol-containing, serine analogues onto gold. Upon subjection to calcium and magnesium, a substantial drop in capacitance was observed. Magnesium displayed the largest influence on the capacitance: a 27% capacitance drop was observed upon introduction of a 1 mM solution of magnesium ions. The lowered capacitance is a result of a change in the potential and charge distribution at the film/electrolyte interface as the cations coordinate to the phosphate groups. Moreover, the relationship between electrode potential and capacitance has been investigated and reveals a significant difference between monovalent and divalent cations. As complementary information, infrared reflection absorption spectra of the phosphorylated monolayer having different counterions are presented. The results reported in this paper indicate that the phosphorylated amino acid analogue monolayers could be used in investigations of the biochemically important coordination of calcium and magnesium to phosphates and phosphorylated amino acids.

Biosensing Techniques↗

Correlation between stinging, TEWL and capacitance.

BACKGROUND/AIMS: To study the correlation between stinging and skin barrier and to make the lactic acid stinging test much safer. METHODS: The modified lactic acid stinging test with 3% and/or 5% aqueous solution of lactic acid combined with transepidermal water loss (TEWL) and capacitance (CAP) was used. The amount of 50 microL-test material was applied on test areas, the skin of both sides nasolabial fold of 50 healthy subjects. Subjects assessed stinging basing on a 4-point scale at 0, 2.5, 5.0 and 8 min TEWL and CAP were measured at baseline and 8 min. RESULTS: The results show that the tendency of decrease in baseline CAP and increase in baseline TEWL are proportional with the increase of the sum of clinical scores (CSS). In 5% lactic acid test, negative correlation between changing ratio of TEWL (PTEWL) and clinical score at 5 min (CS5 min), and negative correlation between changing ratio of capacitance (PCAP) and CS5 min are registered. CONCLUSION: Our data indicate that there is certain relationship between the degree of stinging and the skin barrier. The stinging test method modified by lower concentration aqueous lactic acid and the assessment combined with PTEWL and PCAP is suitable for Chinese to evaluate the skin susceptibility.

Administration, Topical↗

DNA in nanopores: negative capacitance and delta-relaxation at high frequency.

We measured the high frequency dielectric relaxation behavior of DNA molecules confined in nanopores of polycarbonate membrane. The data revealed the existence of a critical frequency omega(c) approximately GHz at which the ac conductivity showed delta-relaxation. Interestingly, the DNA molecules also exhibited a crossover from positive to negative capacitance corresponding to omega(c). The negative capacitance at the critical frequency suggested a strong inductive behavior of DNA molecules in the high frequency regime. The results are interpreted in terms of the confined geometry of the DNA molecules in the nanopores. The interfacial water H-bonded to DNA played a crucial role in determining the high frequency relaxation of DNA molecules. The results indicated that the DNA in nanopores could be designed for application in high frequency bandpass/notch filters.

Computer Simulation↗

Free standing carbon nanotube composite bio-electrodes.

Carbon nanotubes present a new material for the construction of electrodes for electrochemical devices such as batteries, capacitors, and actuators. Such electrodes require high conductivity, strength, and surface area. The latter two requirements are often incompatible. Electrodes composed entirely of carbon nanotubes (bucky paper) have high surface areas but are typically weak, and have insufficient conductivity for practical macroscopic applications. Here we report a technique that uses naturally occurring biopolymers to produce electrodes (free standing films) that exhibit conductivities of 300 S/cm. These composites also have considerable mechanical strength (up to 145 MPa) and sufficient specific capacitance of 19-27 F/g to enable them to be used as freestanding electrodes. One potential application that deserves special attention is that of biocompatible electrodes, where the binder is a biopolymer already used in a range of implants. Preliminary studies reported here show that the new carbon nanotube biopolymer electrodes can foster prolific L929 cell growth.

Animals↗

Purinergic receptors coupled to intracellular Ca2+ signals and exocytosis in rat prostate neuroendocrine cells.

Rat prostate neuroendocrine cells (RPNECs) display a variety of ion channels and exhibit alpha-adrenergic regulation of cytosolic Ca(2+) concentration ([Ca(2+)])(c). In this study, purinergic regulation of [Ca(2+)](c) and exocytosis was investigated in freshly isolated single RPNECs showing chromogranin A immunoreactivity. The presence of P2X and P2Y receptors in RPNECs was verified by the transient activation of Ca(2+)-permeable cationic channels and the release of Ca(2+) from intracellular stores by extracellular ATP, respectively. The transient inward cationic current was effectively activated by alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP) and blocked by 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, suggesting the presence of a P2X(1) or P2X(3) subtype. For the release of stored Ca(2+), ATP and UTP were equally potent, indicating the functional expression of the P2Y(2) or P2Y(4) subtype. The mRNAs for P2X(1) and P2Y(2) were confirmed from reverse transcription-PCR analysis of RPNECs. The application of alpha,beta-MeATP induced large and transient increases in [Ca(2+)](c), which were not attenuated by the blockers of voltage-activated Ca(2+) channels or by depleting intracellular Ca(2+) stores, but were abolished by omitting extracellular Ca(2+). The application of UTP increased [Ca(2+)](c) to 55% of the peak Delta[Ca(2+)](c) induced by alpha,beta-MeATP. The application of alpha,beta-MeATP induced exocytotic responses of RPNECs as monitored by carbon fiber amperometry and capacitance measurements. To our interest, the application of UTP did not induce amperometric currents, but reduced the membrane capacitance, indicating a net endocytosis. From these results, we postulate that a sharp rise in [Ca(2+)](c) by the P2X-mediated Ca(2+) influx is required for exocytosis, whereas the relatively slow release of stored Ca(2+) induces endocytosis in RPNECs.

Adenosine Triphosphate↗

Cytochalasin D attenuates the desensitisation of pressure-stimulated vesicle fusion in guard cell protoplasts.

Fusion of vesicular membranes with the plasma membrane during pressure-driven swelling of guard cell protoplasts was studied using patch clamp capacitance measurements. Hydrostatic pressure pulses were applied via the patch pipette and resulted in an immediate and linear increase in membrane capacitance, a parameter proportional to the surface area. In any given protoplast, pressure-stimulated increases in membrane capacitance could be provoked repetitively. However, the rate of rise in capacitance upon the same strength of stimulation decreased exponentially with time (tau = 4 min) for subsequent pressure stimuli. This process was the result of a desensitisation of the plasma membrane to mechanical forces. Incubation of guard cell protoplasts in cytochalasin D, which depolymerises actin filaments, nearly abolished this desensitisation process. These results suggest that membrane stretch initiates a reactive process that may fortify or stabilise the plasma membrane of guard cell protoplasts.

Actin Cytoskeleton↗

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+.

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.

Animals↗